1. Technical Field
The technical field relates to a humanized anti-human CD34 monoclonal antibody and uses thereof.
2. Description of the Related Art
Human stem cells are a group of undifferentiated cells with the ability to undergo unlimited cell division and differentiation to various cell types, such as hematopoietic stem cells (HSCs). HSCs present in the human marrow and peripheral blood are capable of self-regeneration and differentiation to various cell types. Due to the monocyte-like morphology, HSCs had not been identified until the surface proteins were determined. The surface antigen, CD34, is a transmembrane glycoprotein expressed on the HSC membrane, with an identified molecular weight of about 110 kDa. CD34 has been the first developed biomarker for determining human HSCs (Civin, et al. 1984; Krause, D. S. et al. 1996). Due to the specific binding affinity to the CD34 surface protein, HSCs are capable of being isolated from human blood. The isolated HSCs are available for transplantation in the treatment of several diseases, such as malignant hematologic diseases and solid tumors. However, a success of HSC transplantation therapy raises an issue, how to obtain a sufficient quantity of purified HSCs from the blood (Shpall E J, et al. 1994).
In spite of HSCs, it has been studied that CD34+ stem cells may differentiate to an endothelial type which is called endothelial progenitor cells (EPCs). Several studies have shown that EPCs promote neovascularization and re-endothelialization which correlate to the healing of cardiovascular disorders and wound recovery. Therefore, the way to increase the amount of EPCs or to capture EPCs from the blood is potential for development of medical instruments (Carmen U and Stefanie D, 2004; Jiro A, et al. 2005).
Accordingly, a development of efficient separation and purification of HSCs and EPCs is needed to increase their value in medicine, wherein the antibody-antigen specific binding might be a way to meet the purpose.
Anti-My10 monoclonal antibody (MAb) is a murine-derived antibody against human myeloid cell line KG-1a, which has high affinity and specificity to the CD34 antigen. Several studies have shown that anti-CD34 antibody is a potential MAb for isolation and identification of HSCs (Watt S M, et al., 1987; W. E. Beschorner et al. 1985). In recent researches, murine-derived MAbs have been used in the separation, capture and labeling of HSCs and EPCs. However, murine-derived MAbs may generate other problems. For example, murine-derived MAbs may depart from the isolation column when purifying HSCs from the peripheral blood and enter into the human body with HSC transplantation leading to undesired immune responses.
A major impediment for using murine-derived MAbs in clinical practice is that it may elicit human anti-murine antibody (HAMA) responses in patients (Owens and Young, 1994; Sandhu, 1992; Schroff et al., 1985). Hence, to improve efficiency in clinical use, genetic engineering technology has been employed to replace the murine content with the amino acid residues of human counterparts, which reduces the possibility of inducing immunogenicity in patients.
An ideal for antibody humanization is that it should be capable of maintaining specificity and affinity toward an antigen and reduce immunogenicity as much as possible. So far, many approaches have been used for antibody humanization, such as chimeric antibodies, which consists of murine antigen-binding variable regions fused genetically to human antibody constant regions. This was the earliest attempt to reduce immunogenicity (Morrison et al., 1984). However, chimeric antibodies would still generate undesirable anti-variable region responses (Bruggemann et al., 1989).
CDR-grafting is another approach involving the transfer of the complementarity determining regions (CDRs) from a rodent antibody to the Fv frameworks (FRs) of a human antibody (Verhoeyen et al., 1988). Unfortunately, the interface changes between CDRs and new FRs may largely disturb the binding to the antigen. The initial CDR-grafted antibodies tend to lose parental binding affinity, and therefore require additional work for back-mutation of several murine framework amino acids, which are regarded as crucial for CDR loop conformations. Thus, the method is not only time-consuming but results in a low probability of success.
As the development of structural biology, the murine antibody can be constructed in a three-dimensional structure on homology modeling and substitute the amino acid residues that possibly hinder the antigen binding in the murine Fv frameworks with the corresponding residues of human antibodies. This method maintains the advantages of humanization and eliminates HAMA responses, becoming a trend for antibody humanization.